The focus of this project is to explore the clinical significance of our understanding of the origin of neuronal and glial cells. For several years, our group has been at the leading edge of transplantation into the nervous system. As our ability to isolate and culture cells has improved, we have been able use the improved definition of the donor cells with animal models of disease. In the past year,we made an important contribution by showing that we can obtain differentiated oligodendrocytes in animals from glial precursors obtained from embryonic stem (ES) cells. The isolation of human ES (and the closely related EG) cell lines has generated considerable interest in the idea that these cells lines can be used to provide a constant supply of replacement cells to treat human disease. By definition, ES cells can generate all the cells of the animal but it was not clear how easy it would be in practice to obtain large numbers of highly enriched specific somatic precursor from ES cells. Our work suggests that this can be done relatively simply. In this particular case our study is relevant to demyelinating diseases, such as multiple sclerosis. But the significance of the work is more general as it is one of the first demonstrations that a specific somatic precursor cell can be obtained from ES cells.A second major focus in this project follows from our recent discovery of stem cells that generate dopamine-producing neurons. The systematic access to large numbers of these neurons allowed by stem cell technology promises new tools to analyze the mechanisms controlling the birth and death of these important cells. This insight can then be used to develop new cell and pharmacological therapies for Parkinson?s disease.While we have demonstrated that stem cells for dopamine neurons can be expanded, we cannot provide the large numbers needed for a clinical grafting or drug screening program. Initially, we were disappointed on the limit of the number of dopamine neurons but with more work we can see that this problem can be solved. We can change the extracellular conditions for growing the stem cell and gain considerable increase in the efficiency of differentiation to the dopamine-producing fate. For example, in collaboration with a group at Cal Tech, we have shown that the oxygen concentration is a critical feature of the culture. In addition, we have taken a systematic approach to analyze changes in gene expression as the stem cells are expanded. This allows us to replace genes that are down-regulated as stem cells expand. Restoring gene expression from introduced cDNAs restores features of the dopaminergic fate. Both of these approaches overcome the limit to the numbers of dopaminergic neurons. But they do something else that may be even more important, i.e., they give us a new understanding of the mechanisms that control differentiation of the cell of interest. The mechanisms controlling the differentiation of the dopaminergic cell may provide new strategies to treat Parkinson?s disease by manipulating these pathways. You may say that these events that are only important in the differentiation of the cells but there is increasing evidence that these control systems may still be active in the adult.In the context of providing large numbers of cells for grafting, we have already discussed the importance of controlling the birth of neurons. It is also clear that we must define the causes of neuronal death. The architecture of the brain is normally formed by neuronal death. This is often called programmed cell death. Neurons (or parts of neurons, synapses) must die to establish the normal cell numbers and connections in the brain. This loss is appropriately limited but when it is not adequately controlled, leads to neurodegenerative disease. What normally regulates the death of neurons? Neurons that make good contact with their targets are thought to receive life- giving rewards from their partners. This kind of interaction has rarely been studied but we have developed simple experimental systems to demonstrate the control systems that regulate synpase function and neuronal survival. This was first set up for hippocampal and cortical neurons; the specific details of these cycles may be relevant to Alzheimer?s disease where there is a loss of synapses followed by a loss of neurons. However, we believe that the same approach can be applied to neurons in other brain regions such as the striatum and mesencephalon where the applications to Huntington?s and Parkinson?s diseases will be pursued.